Pharmaceutical application of 15- or 16-substituted testosterone analogues

ABSTRACT

The invention relates to pharmaceutical dosage units for oral, transmucosal or transdermal administration containing 15- or 16-substituted testosterone analogues, as well as to therapeutic methods that employ these testosterone analogues. More particularly, the invention is concerned with such pharmaceutical dosage units containing at least 10 μg of an androgenic steroid selected from the group consisting of 15-hydroxytestosterones, 16-hydroxytestosterones, precursors thereof and mixtures of these hydroxytestosterones and/or their precursors; and a pharmaceutically acceptable excipient. The term “15-hydroxytestosterones” encompasses both 15α-hydroxytestosterone (15α, 17β-dihydroxy-4-androsten-3-one) and 15β-hydroxytestosterone (15β, 17β-dihydroxy-4-androsten-3-one). Similarly, the term “16-hydroxytestosterones” encompasses both 16α-hydroxytestosterone hydroxytestosterone (16α, 17β-dihydroxy-4-androsten-3-one) and 16β-hydroxytestosterone (16β, 17β-dihydroxy-4-androsten-3-one). The androgenic steroids according to the invention are advantageously employed in e.g. a method of treating or preventing androgen deficiency or a method of hormonal contraception.

TECHNICAL FIELD

The present invention is in the field of steroid chemistry. Moreparticularly it relates to pharmaceutical dosage units containing 15- or16-substituted testosterone analogues, as well as to therapeutic methodsthat employ these testosterone analogues. The testosterone analogues ofthe invention possess androgenic activity.

BACKGROUND OF THE INVENTION

British patent GB-B 774,064 is concerned with a process for theproduction of 15-substituted testosterone, particularly15-hydroxytestosterone. Therapeutic applications of these substances arenowhere mentioned in the patent.

French patent application FR-A 2 035 786 describes veterinaryapplications of 16α-α-D-glucosiden of 16α,17-dihydroxysteroids. It isstated that these steroids can be used as estrogens to prevent ovulationin animals such as rodents, dogs, cow and sheep. Claim 3 mentions theglucoside of 16α-hydroxytestosterone. Only parenteral administration ismentioned in the French application.

At present, the main therapeutic application of androgenic steroids isin the treatment of hypogonadal males. Methods of treating androgendeficiency comprising the administration of androgens, such astestosterone, dihydrotestosterone, dehydroepiandrosterone and variousesters of testosterone, or derivatives and analogues such as mesteroloneare known in the art.

Three types of androgen deficiency in males are usually distinguished,i.e. primary androgen deficiency (testicular insufficiency), secondaryandrogen deficiency (hypothalamo-hypophyseal insufficiency) and androgendeficiency in ageing males (ADAM), also known as “male menopause” or“andropause”.

As regards the long-term administration of androgens to males, adistinction can be made between therapy and supplementation. Therapytypically requires relatively high doses that are usually similar to therate of production of endogenous androgens. Supplementation on the otherhand is suitably done with dosages that are below the rate of productionof endogenous androgens (i.e. testosterone, dihydrotestosterone anddehydroepiandrosterone).

Because of concerns about undesirable side effects, androgens are onlyused sparingly in both therapy and hormone supplementation. Indeed,androgens are normally only used for therapy in human males when primaryor secondary androgen deficiency has been diagnosed.

Only a few androgens, e.g. dehydroepiandrosterone (DHEA) and17α-alkylated derivatives of testosterone, are suitable for oraladministration because, unlike testosterone, they are largely resistantto hepatic metabolism. However, disadvantages of oral to administrationare associated with the bad absorption of these androgens and therelatively high effect they exert on the liver and particular the livermetabolism (Bhasin et al. (1997) J. Clin. Endocr. Metab. 82:3-8). Thisis why, in existing protocols, androgens are generally administered inthe form of 2-3 weekly depot injections or implants.

It has also been suggested in the prior art to use a combination of aprogestogen and an androgen in a method of male contraception. In such amethod the progestogen is administered in a sufficiently high amount tohalt spermatogenesis—leading to azoospermia—and the androgen isco-administered to prevent androgen deficiency which would otherwiseresult from the administration of the progestogen. Because of concernsabout reliability and possible side-effects of the androgenic component,particularly when administered orally, male contraceptive have not yetmade it beyond the experimental stage.

Other therapeutic uses of androgens that have been proposed in the priorart include treatment of the wasting syndrome and retro-viral druginduced lipodystrophia in HIV infected individuals, enhancement ofrecovery of critically ill catabolic individuals, treatment of benigngynaecological disorders hormonal contraception in females, delayedpuberty, female-to-male conversion.

As will be apparent from the above, there is an unmet need for androgensthat (i) can be used effectively in the above mentioned therapeuticmethods without causing undesirable side-effects, (ii) produce a veryconsistent, i.e. predictable, impact and/or (iii) may be administered ina convenient manner, especially orally.

SUMMARY OF THE INVENTION

The inventors have unexpectedly discovered that steroids which have notbeen used in therapeutic applications, i.e. 15-hydroxy or 16-hydroxysubstituted testosterone analogues, meet the aforementionedrequirements.

Surprisingly, it was found that the hydroxytestosterone analoguesaccording to the present invention combine adequate androgenic potencywith acceptable oral bioavailability and minimum impact on livermetabolism. Consequently, the present androgens are particularly suitedfor any therapeutic applications for which androgens have been employedor recommended. The present hydroxytestosterone analogues may also bedelivered effectively, particularly in relatively low dosages such asthose typically required by females, by transmucosal or transdermaladministration.

Another advantage of the hydroxytesterone analogues according to theinvention is their predictable and consistent therapeutic impact, whichis believed to be related to the efficiency of uptake, their low hepaticeffect and/or their low affinity for sex hormone binding globulin(SHBG). In contrast, the therapeutic effect of known androgens that aresometimes administered orally, notably DHEA or 17α-alkylated derivativesof testosterone, is very dependent on an individual's physiology andeven his/her diet. Consequently, these androgens are usually applied inrelatively high dosages in order to ensure that the minimum effectivedosage is achieved in each individual. Naturally such high dosages havethe disadvantage that they lead to relative overdosing in someindividuals, which in turn is likely to produce pronounced side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of competition curves showing displacement of [³H]DHTfrom human SHBG using 5α-dihydrotestosterone (DHT), testosterone (T),dehydroepiandrosterone (DHEA), 15α-hydroxytestosterone (15α-OH-T),15β-hydroxytestosterone (15β-OH-T) as competitors. Results are expressedas bound (B) cpm at each competitor concentration/total (T) cpm bound inabsence of competitor as a percentage.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, one aspect of the invention relates to a pharmaceuticaldosage unit for oral, transmucosal or transdermal administrationcontaining at least 10 μg of an androgenic steroid selected from thegroup consisting of 15-hydroxytestosterones, 16-hydroxytestosterones,precursors thereof and mixtures of these hydroxytestosterones and/ortheir precursors; and a pharmaceutically acceptable excipient.

The term “15-hydroxytestosterones” encompasses both15α-hydroxytestosterone (15α,17β-dihydroxy-4-androsten-3-one) and15β-hydroxytestosterone (15β,17β-dihydroxy-4-androsten-3-one).Similarly, the term “16-hydroxytestosterones” encompasses both16α-hydroxytestosterone hydroxytestosterone(16α,17β-dihydroxy-4-androsten-3-one) and 16β-hydroxytestosterone(16β,17β-dihydroxy-4-androsten-3-one).

The term “precursor” as used throughout this document refers tosubstances which are converted into one of the hydroxytestosterones ofthe invention following the administration of said precursor to a humansubject.

Preferred precursors of the androgenic steroids according to the presentinvention are derivatives of the present hydroxytestosterones whereinthe hydrogen atom of at least one hydroxylgroup has been substituted byan acyl radical of a hydrocarbon carboxylic, sulfonic or sulfamic acidof 1-25 carbon atoms; tetrahydrofuranyl; tetrahydropyranal; or astraight or branched chain glycosydic residue containing 1-20 glycosidicunits per residue.

It has been shown that the hydroxytestosterones of the present inventionoccur in mammals, which means that they offer the important advantagethat they can be labelled as natural (Yamazaki et al. Arch BiochemBiophys 1997 Oct. 1; 346(1):161-9). In addition, as can be derived fromthe aforementioned article, both 15α-hydroxytestosterone,15β-hydroxytestosterone and 16β-hydroxytestosterone occur in the humanbody, which means that prima facie the risk of undesired side-effects islower than is the case for synthetic androgens or natural androgens thatare not found in the human body. Thus, in a particularly preferredembodiment of the present invention, the androgenic steroid is selectedfrom the group consisting of 15α-hydroxytestosterone,15β-hydroxytestosterone, 16β-hydroxytestosterone, precursors thereof andmixtures of these hydroxytestosterones and/or their precursors. Evenmore preferably, the androgenic steroid is selected from the groupconsisting of 15α-hydroxytestosterone, 15β-hydroxytestosteroneprecursors thereof and mixtures of these hydroxytestosterones and/ortheir precursors.

The present oral dosage units may be prepared according to any methodknown to the art for the manufacture of pharmaceutical compositions andsuch units may contain one or more agents selected from the groupconsisting of sweetening agents, flavouring agents, colouring agents andpreserving agents. Said dosage units may suitably contain a non-toxicpharmaceutically acceptable excipient. In case of e.g. tablets, theseexcipients may be, for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, such as maize starch,or alginic acid; binding agents, such as starch, gelatine or acacia; andlubricating agents, such as magnesium stearate, stearic acid or talc.Tablets may be uncoated or may be coated by known techniques to delaydisintegration and adsorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monostearate or glyceryl distearatealone or with a wax may be employed.

Examples of dosage unit forms that may be administered by oral routeinclude tablets, soft gelatine capsules, including solutions used insoft gelatine capsules, aqueous or oil suspensions, emulsions, pills,lozenges, troches, syrups, elixirs and the like. Formulations for oraluse may also be presented as hard gelatine capsules wherein the activeingredient is mixed with an inert solid diluent, for example calciumcarbonate, calcium phosphate or kaolin, or as soft gelatine capsuleswherein the steroid component is mixed with water or an oil medium, suchas peanut oil, liquid paraffin or olive oil. In a particularly preferredembodiment, the oral dosage units according to the present invention areprovided in the form of solid or semi-solid dosage units, especially inthe form of tablets, capsules, cachets, pellets, pills, powders orgranules.

Aqueous suspensions according to the invention contain the steroids in amixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Ophthalmic formulations, as is known inthe art, will be adjusted for osmotic pressure.

Oil suspensions may be formulated by suspending the steroid component ina vegetable oil, such as arachide oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oil suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water may beformulated from the steroid component in a mixture with a dispersing,suspending and/or wetting agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose disclosed above. Additional excipients, for example sweetening,flavouring and colouring agents, may also be present.

The pharmaceutical dosage unit of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachide oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifying agents includenaturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavouring or a colouring agent.

The steroids of this invention may also be administered in the form ofsuppositories for rectal or vaginal administration of the drug. Thesedosage units can be prepared by mixing the steroid with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperatures and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

The present dosage unit preferably contains at least 20 μg, morepreferably at least 40 μg and most preferably at least 60 μg of theandrogenic steroid. In case the dosage unit is meant to provide asustained release of the androgenic steroid, e.g. in case of atransdermal patch, the amount of androgenic steroid contained in thedosage unit may be as high as 1000 mg. Preferably the amount of theandrogenic steroid does not exceed 500 mg, more preferably it does notexceed 200 mg. Most preferably the amount of androgenic steroid in thedosage unit does not exceed 100 mg.

In a particularly preferred embodiment of the invention, the dosage unitis designed for oral administration. Examples of oral dosage units whichmay suitably be used to deliver the present steroids include tablets,capsules, cachets, pellets, pills, powders or granules.

Typically the present pharmaceutical oral dosage unit has a weight inthe range of 0.1-10 grams. The amount of androgenic steroid contained inthe oral dosage unit is preferably at least 30 μg, more preferably atleast 50 μg and most preferably at least 80 μg. Usually the amount ofsteroid contained in the oral dosage unit will not exceed 50 mg, morepreferably it will not exceed 30 mg, most preferably it will not exceed20 mg.

The hydroxytesterone analogues of the present invention mayadvantageously be to employed in a method of male contraception. Asmentioned herein before, such a method requires the combinedadministration of a progestogen and an androgen. Typically the amount ofprogestogen included in the present dosage unit will exceed 10 μg,preferably it will exceed 30 μg. In a particularly preferred embodiment,the present dosage unit additionally contains a progestogen in an amountequivalent to between 75 and 800 μg, more preferably between 100 and 500μg levonorgestrel.

In other applications, e.g. in female hormone replacement therapy orfemale contraception, it may be advantageous to administer the presenthydroxytestosterone analogues in combination with an estrogen.Accordingly, the present dosage unit, in addition to the presentandrogenic steroid, may suitably contain an estrogen, preferably in anamount of at least 10 μg, more preferably in an amount of at least 15μg.

Examples of progestogens that may employed in the present dosage unit aswell as in the methods described herein include progesterone,levonorgestrel, norgestimate, norethisterone, dydrogesterone,drospirenone, 3-beta-hydroxydesogestrel, 3-keto desogestrel(=etonogestrel), 17-deacetyl norgestimate, 19-norprogesterone,acetoxypregnenolone, allylestrenol, anagestone, chlormadinone,cyproterone, demegestone, desogestrel, dienogest, dihydrogesterone,dimethisterone, ethisterone, ethynodiol diacetate, fluorogestoneacetate, gastrinon, gestodene, gestrinone, hydroxymethylprogesterone,hydroxyprogesterone, lynestrenol (=lynoestrenol), medrogestone,medroxyprogesterone, megestrol, melengestrol, nomegestrol, norethindrone(=norethisterone), norethynodrel, norgestrel (includes d-norgestrel andd1-norgestrel), norgestrienone, normethisterone, progesterone,quingestanol,(17alpha)-17-hydroxy-11-methylene-19-norpregna-4,15-diene-20-yn-3-one,tibolone, trimegestone, algestone acetophenide, nestorone, promegestone,17-hydroxyprogesterone esters, 19-nor-17hydroxyprogesterone,17alpha-ethinyl-testosterone, 17alpha-ethinyl-19-nor-testosterone,d-17beta-acetoxy-13beta-ethyl-17alpha-ethinyl-gon-4-en-3-one oxime,precursors of these compounds that are capable of liberating theseprogestogens in vivo when used in the present method and combinationsthereof. Preferably the progestogen is selected from the groupconsisting of progesterone, desogestrel, etonogestrel, gestodene,dienogest, levonorgestrel, norgestimate, norethisterone, drospirenone,trimegestone, dydrogesterone, precursors of these progestogens andcombinations thereof.

Examples of estrogens that may suitably be used in accordance with thepresent invention include ethinyl estradiol, mestranol, quinestranol,estradiol, estrone, estran, estriol, estetrol, conjugated equineestrogens, precursors thereof that are capable of releasing such anestrogen in vivo when used in the present method and combinationsthereof. Preferably the estrogen is selected from the group consistingof ethinyl estradiol, estradiol, estetrol and combinations thereof.

Preferred precursors of the androgenic steroids according to the presentinvention are derivatives of the present hydroxytestosterones whereinthe hydrogen atom of at least one hydroxylgroup has been substituted byan acyl radical of a hydrocarbon carboxylic, sulfonic or sulfamic acidof 1-25 carbon atoms; tetrahydrofuranyl; tetrahydropyranal; or astraight or branched chain glycosydic residue containing 1-20 glycosidicunits per residue.

Another aspect of the invention relates to a method of curatively orprophylactically treating a mammal, said method comprising oral,transmucosal or transdermal administration to said mammal of the presentpharmaceutical dosage unit as described herein before. The termtransmucosal administration as used herein, refers to intravaginal,intra-uterine, rectal, intranasal, pulmonary, buccal and sublingualdelivery, intravaginal delivery being most preferred. The present methodis particularly suitable for treating humans.

Oral, rectal, intranasal, buccal and pulmonary administration areideally suited for (at least) once daily administration. Transdermaladministration is advantageously applied at frequencies between once aday and once a month. Intravaginal and intra-uterine administrations areadvantageously operated at administration frequencies between onceweekly and once monthly.

For reasons of convenience and also to achieve high compliance rates,the present method preferably utilises administration intervals of 1day, 1 week or 1 month. Regimens that employ once daily oraladministration, once weekly transdermal administration or once monthlyintravaginal administration are particularly preferred. Most preferablythe present method comprises at least once daily administration.

The androgenic steroid(s) of the invention are advantageouslyadministered in an average daily amount which is at least 0.5 μg per kgof bodyweight. Preferably the average daily amount is at least 1 μg perkg of bodyweight, more preferably at least 1.5 μg per kg of bodyweight.The average daily amount usually does not exceed 1.5 mg per kg ofbodyweight. Preferably the average daily amount does not exceed 1 mg perkg of bodyweight, more preferably it does not exceed 0.5 mg per kg ofbodyweight.

The method according to the present invention is preferably employed ina method of treating or preventing androgen deficiency (in males orfemales); a method of hormonal contraception (in males or females); amethod of treating or preventing wasting syndrome, anti-retroviral druginduced lipodystrophia, lack of well-being or fatigue in HIV infectedindividuals; a method of reversing catabolic state caused by a chronicillness, surgical intervention, oncological condition, trauma and/ormalnutrition; a method of treating or preventing leydig cell dysfunctionand germinal epithelial damage following cytotoxic chemotherapy; amethod of treating or preventing fatigue or maintaining weight,hemoglobine or neutrophil count during or subsequent to cytotoxicchemotherapy or radiotherapy; a method of treating or preventing benigngynaecological disorders; a method of improving libido (in males orfemales); a method of treating or preventing delayed puberty; or amethod of supporting female-to-male conversion. As mentioned hereinbefore, the administration of the present steroids is particularlyadvantageous when employed in a method of treating or preventingandrogen deficiency, a method of hormonal contraception or a method oftreating or preventing benign gynaecological disorders. It is noted thatsymptoms of androgen deficiency can occur in both males and females. Ina particularly preferred embodiment the present is used to treat orprevent androgen deficiency or employed as a contraceptive method inmales.

The aforementioned methods usually employ continuous administration ofthe present steroids during a period of at least 10 days, preferably ofat least 20 days. In a particular preferred embodiment the presentsteroids are administered during a period of at least 100 days withadministration-free intervals that do not exceed 10 days, preferably donot exceed 8 days.

In a preferred embodiment, the present method comprises theco-administration of a progestogen in an average daily amount of atleast 0.2 μg per kg of bodyweight and/or an estrogen in an average dailyamount of at least 0.2 μg per kg of bodyweight. Typically, the averagedaily amount of co-administered progestogen or estrogen will not exceed1 mg per kg of bodyweight, more preferably said amount does not exceed500 μg per kg of bodyweight, most preferably it does not exceed 300 μgper kg of bodyweight.

Yet another aspect of the invention relates to the use of an androgenicsteroid selected from the group consisting of 15-hydroxytestosterones,16-hydroxytestosterones, precursors thereof and mixtures of thesehydroxytestosterones and/or their precursors in the preparation of anoral, transmucosal or transdermal dosage unit as described herein. In aparticularly preferred embodiment, said androgenic steroid is used inthe preparation of a solid or semi-solid oral dosage unit, especially anoral dosage unit in the form of a tablet, a capsule, a cachet, a pellet,a pill, a powder or granules.

The invention is further illustrated by means of the following examples.

EXAMPLES Example 1

An established competitive steroid binding assay was used to determinethe relative binding affinity of 15α-hydroxytestosterone(15α,17β-dihydroxy-4-androsten-3-one), 15β-hydroxytestosterone(15β,17β-dihydroxy-4-androsten-3-one) and 16β-hydroxytestosterone(16β,17β-dihydroxy-4-androsten-3-one) to the androgen receptor (AR).Testosterone (17β-hydroxy-4-androsten-3-one) and5α-dihydroxytestosterone (DHT, 5α,17β-dihydroxy-androstan-3-one) servedas controls in this assay.

The method employed was adapted from the scientific literature anddescribed in detail by Chang et al (1987, J. Steroid Biochem., 27, 1-3,123-131). Recombinant rat AR was expressed and purified from E. Coli.The in vitro assays involved the use of AR and [³H]mibolerone, at afixed concentration of 1.5 nM, as the labeled ligand. Recombinant AR wasdissolved in binding buffer (50 mM Tris-Base, pH 7.5, 0.8 mM NaCl, 10%glycerol, 2 mM dithiothreitol, 1 mg/ml BSA and 2% ethanol) and duplicatealiquots were then incubated with [³H]mibolerone at a finalconcentration of 1.5 nM, together with a vehicle control (1.0% DMSO), orthe same amount of vehicle containing increasing concentrations ofunlabeled steroid ligands as competitors. After incubation for 4 h at 4°C., the unbound ligands were removed and the amounts of [³H]miboleronebound to the AR was measured. The average amounts of [³H]miboleronebound to AR at each concentration of competitor were used to makeinhibition curves. IC50 values were subsequently determined by anon-linear, least squares regression analysis. Inhibition constants (Ki)were calculated using the equation of Cheng and Prusoff (Cheng et al.,1973, Biochem. Pharmacol., 22, 3099-3108), using the measured IC50 ofthe tested compounds, the concentration of radioligand employed in theassay, and the historical values for the Kd of the radioligand, whichhas been established as 3 nM.

Mean Ki values obtained for 15α-hydroxytestosterone,15β-hydroxytestosterone, 16β-hydroxytestosterone, testosterone and DHTfrom three separate experiments are shown in Table 1. For comparision ofbinding affinities, the Ki value of DHT (which shows the highestaffinity) was arbitrarily set at 100% and used to calculate the relativeaffinities (Table 1). As compared to DHT and testosterone,15α-hydroxytestosterone, 15β-hydroxytestosterone and16β-hydroxytestosterone bind with 2 to 3 orders of magnitude lessaffinity to AR (Table 1).

TABLE 1 Experimentally determined inhibition constants (Ki) for ARbinding of 15α-hydroxytestosterone, 15β-hydroxytestosterone,16β-hydroxytestosterone, testosterone and 5α-dihydroxytestosterone(DHT). Relative binding affinities are also shown, using Ki value of DHTas 100%. AR-binding Relative Ki affinity Trivial Name (Chemical Name)(nM) (%) 15α-hydroxytestosterone 1820 0.05 (4-ANDROSTEN-15α,17β-DIOL-3-ONE) 15β-hydroxytestosterone 557 0.18 (4-ANDROSTEN-15β,17β-DIOL-3-ONE) 16β-hydroxytestosterone 814 0.12 (4-ANDROSTEN-16β,17β-DIOL-3-ONE) Testosterone 2 50 (4-ANDROSTEN-17β-OL-3-ONE)5α-dihydroxytestosterone, DHT 1 100 (5α-ANDROSTAN-17β-DIOL-3-ONE)

Example 2

To determine the in vivo androgenic potency of 15-substitutedtestosterone derivatives, 15α-hydroxytestosterone(15α,17β-dihydroxy-4-androsten-3-one) and 15β-hydroxytestosterone(15β,17β-dihydroxy-4-androsten-3-one) were administered orally, oncedaily, to immature male Wistar rats for 5 consecutive days. Testosterone(17β-hydroxy-4-androsten-3-one) and 5α-dihydroxytestosterone (DHT,5α,17β-dihydroxy-androstan-3-one), both administered orally, served aspositive controls in this bioassay.

15α-hydroxytestosterone, 15β-hydroxytestosterone, testosterone and5α-dihydroxytestosterone were dissolved in an aqueous suspension of 2%Tween 80, at final test concentrations of 3 mg/ml and 1 mg/ml. Groups of5 immature male Wistar rats, weighing 50±2 grams, received eithervehicle treatment or were administered 30 or 10 mg/kg/day test substanceby oral gavage in a dosing volume of 10 ml per kg for 5 consecutivedays. During the experiment, animals were housed in cages of 45×23×15 cmand maintained in a controlled temperature of 22-24° C. and humidity(60-80%) environment with 12 hours light/dark cycles. Food (lab diet,rodent diet, PMI nutrition international) and water intake was adlibitum.

The animals were sacrificed 24 hours after receiving the final dose andthe wet weight of the seminal vesicle of each animal was recorded andaverage wet weight per group was calculated. Increase in average seminalvesicle wet weight relative to the average seminal vesicle wet weight ofvehicle treated rats was considered indicative for in vivoandrogenicity.

In vivo androgenicity data are shown in Table 2. Both 15α and 15βhydroxy-substituted analogues of testosterone induced a dose-dependentand significant increase in seminal vesicle wet weight. These in vivoandrogenic effects were similar or equipotent to the dose-dependenteffects observed for testosterone and considerably more potent than thein vivo androgenicity observed after oral administration of DHT, aclassical androgen and the active metabolite of testosterone in vivo(O'Donnel et al., 1996, Endocrinology, 137, 2703-2710).

TABLE 2 Average increase in seminal vesicle wet weight relative to theaverage wet weight of vehicle (2% Tween-80) treated animals for groupsof 5 immature Wistar rats, treated once daily, orally, for fiveconsecutive days with 15α-hydroxytestosterone, 15β-hydroxytestosterone,testosterone or 5α-dihydroxytestosterone (DHT). Relative androgenicpotencies are also shown, using DHT as reference (100%). In vivoandrogenicity Increase seminal Relative Oral dose vesicle wet potencyTrivial Name (Chemical Name) mg/kg/day weight (%) (%)15α-hydroxytestosterone 30 40 148 (4-ANDROSTEN-15α, 10 28 56017β-DIOL-3-ONE) 15β-hydroxytestosterone 30 35 130 (4-ANDROSTEN-15α, 1020 400 17β-DIOL-3-ONE) Testosterone 30 47 174 (4-ANDROSTEN-17β-OL-3-ONE)10 41 820 5α-dihydroxytestosterone, 30 27 100 DHT 10 5 100(5α-ANDROSTAN-17β-DIOL- 3-ONE)

Example 3

An established competitive steroid-binding assay (Hammond andLahteenmaki. 1983. Clin Chem Acta 132:101-110) was used to determine therelative binding affinity of 15α-hydroxytestosterone (15α-OH-T),15β-hydroxytestosterone (15β-OH-T), dehydroepi-androsterone (DHEA),testosterone (T) and 5α-dihydrotestosterone (DHT) for human sex HormoneBinding Globulin (SHBG).

Human SHBG was purified from transgenic mouse serum, as describedpreviously (Avvakumov G V et al., 2000. J Biol Chem 275: 25920-25925).The human SHBG prepared in this way was assessed to be >99% pure bypolyacrylamide gel electrophoresis under denaturing conditions. Itssteroid-binding characteristics are indistinguishable from SHBG in humanserum (Avvakumov G V et al., 2000. J Biol Chem 275: 25920-25925). The invitro assay involved the use of the purified human SHBG and [³H]DHT asthe labeled ligand. Human SHBG was treated for 30 min at roomtemperature with a dextran-coated charcoal (DCC) suspension in phosphatebuffered saline (PBS) to remove any steroid ligand. After centrifugation(2,000×g for 10 min) to sediment the DCC, the supernatant containing thehuman SHBG was diluted in PBS to a concentration of 1 nM based on itssteroid binding capacity.

Duplicate aliquots (100 μl) of this human SHBG solution were thenincubated with an equal volume of [³H]DHT at 10 nM, together with 100 μlof PBS alone or the same amount of PBS containing increasingconcentrations of unlabeled steroid ligands as competitors inpolystyrene test tubes. After incubation for 1 h at room temperature thereaction mixtures were placed in an ice bath for a further 15 min.Aliquots (600 μl) of an ice cold suspension of DCC were then added toeach tube, and after a brief 2 seconds mixing, each tube was incubatedin an ice bath for 10 min. The unbound ligands adsorbed to DCC were thenremoved by centrifugation (2,000×g for 15 min at 4° C.), and the amountsof [³H]labeled DHT bound to SHBG were counted in 2 ml ACS scintillationcocktail using in liquid scintillation spectrophotometer. The averageamounts of [³H] labeled DHT bound to SHBG at each concentration ofcompetitor (B) were expressed as a percentage of the average amounts of[³H]labeled DHT bound to SHBG in the absence of competitor (T), and wereplotted against the concentration of competitor in each assay tube.

The results of the competitive binding assays are depicted in FIG. 1.The data show that 15α-OH-T and 15β-OH-T do not bind human SHBG, whencompared to DHT, T or DHEA in a competitive binding assay in which[³H]DHT was used as labeled ligand. Therefore, in contrast to otherandrogens, the binding of 15α-OH-T or 15β-OH-T to human SHBG can beconsidered negligible.

1. A pharmaceutical oral dosage unit containing at least 10 μg of anestrogen and at least 10 μg of a steroid selected from the groupconsisting of 15-hydroxytestosterones, precursors thereof, mixturesthereof and precursors of said mixtures; and a pharmaceuticallyacceptable excipient, wherein said oral dosage unit is selected from thegroup consisting of a tablet, a capsule and a chachet, wherein theprecursors of the hydroxytestosterones are derivatives of thehydroxytestosterones wherein a hydrogen atom of at least one hydroxylgroup has been substituted by an acyl radical of a hydrocarboncarboxylic, sulfonic or sulfamic acid of 1-25 carbon atoms;tetrahydrofuranyl; tetrahydropyranal; or a straight or branched chainglycosidic residue containing 1-20 glycosidic units per residue, andwherein the pharmaceutical oral dosage unit is orally active.
 2. Thepharmaceutical oral dosage unit according to claim 1, wherein thesteroid is selected from the group consisting of15α-hydroxytestosterone, precursors thereof, mixtures of15α-hydroxytestosterone, and precursors of these mixtures of thesehydroxytestosterones.
 3. The pharmaceutical oral dosage unit accordingto claim 1, wherein the steroid is selected from the group consisting of15β-hydroxytestosterone, precursors thereof, mixtures of15β-hydroxytestosterone, and precursors of these mixtures of thesehydroxytestosterones.
 4. The pharmaceutical oral dosage unit accordingto claim 1, wherein the oral dosage unit is a tablet.
 5. Thepharmaceutical oral dosage unit according to claim 1, wherein the oraldosage unit contains between 20 μg and 1000 mg of the androgenicsteroid.
 6. The pharmaceutical oral dosage unit according to claim 1,wherein the oral dosage unit additionally contains at least 10 μg of aprogestogen.
 7. A method of hormone replacement therapy in a femalemammal, said method comprising oral administration to said mammal of theoral dosage unit according to claim
 1. 8. The method according to claim7, wherein the method comprises the administration of the steroid in anaverage daily amount in the range of 0.5 μg to 1.5 mg per kg ofbodyweight.
 9. A method of contraception in a female mammal, said methodcomprising oral administration to said mammal of the oral dosage unitaccording to claim
 1. 10. The method according to claim 9, wherein themethod comprises the administration of the steroid in an average dailyamount in the range of 0.5 μg to 1.5 mg per kg of bodyweight.